A-fucosylation detection in antibodies

ABSTRACT

This invention describes a new analytical method to determine the quantity and distribution of fucose per Fc within an antibody preparation.

This invention relates to a method for detecting the presence or absenceof fucose residues within a glycosylated antibody or a fragment thereof.

BACKGROUND

While the variable regions within the Fab (fragment antigen binding)domains of antibodies are responsible for the recognition of theantigen, the Fc (fragment crystallizable) region represents an invariantpart of the antibody that is responsible for the mediation of effectorfunctions. In the case of immunoglobulin G (IgG) these encompass thefixation of complement and the binding to Fcγ receptors (FcγRs). Thepresence of an N-linked oligosaccharide at a single conserved site(Asn297) within the CH2 domain of the homodimeric Fc fragment ismandatory for the mediation of both of these effector functions. It wasonly recently discovered that modification of the attached carbohydratescan also have an affinity improving effect for the interaction betweenFcγRIIIa and IgG. The carbohydrate modification responsible for thiseffect is the absence of a fucose residue which is usually attached tothe first N-acetylglucosamine (GlcNAc) residue in the biantennarycomplex-type IgG glycan (FIG. 1).

It could be demonstrated by in vivo and in vitro experiments that suchincreased affinity results in enhanced antibody-dependent cellularcytotoxicity (ADCC) mainly mediated by natural killer (NK) cells.Consequently, it is also believed that such a-fucosylated antibodieshave an improved efficacy in treatments that aim to eradicate opsonizedcells.

The generation of a-fucosylated antibodies represents an importantbiotechnological challenge which can be achieved by several methods.While cell lines with a complete depletion of enzymes involved in thebiosynthesis of fucosylation (e.g. by gene knockout) may yieldquantitatively a-fucosylated antibodies, most other methods do not. Forexample, siRNA treatment or co-cultivation of antibody-expressing cellswith kifunensine (Zhou et al., Biotechnol Bioeng (2008) 99, 652-665), aswell as carbohydrate modification by N-acetylglucosaminyltransferase III(GnT-III), which promotes the formation of bisected oligosaccharidesconsequently inhibiting the fucosylation reaction (Umana et al., NatBiotech (1999) 17, 176-180), lead to only partially a-fucosylatedantibodies.

These partially a-fucosylated antibodies can principally exhibit aheterogeneous a-fucosylation distribution within a pool of antibodies.For example, fucosylation rates can be different during fermentation.Also, the event of fucosylation could be cooperative, i.e. the secondfucosylation on the homodimeric antibody may occur with an increased(positive cooperativity) or decreased (negative cooperativity) ratecompared to the first one.

The FcγRIIIa/IgG complex has a 1:1 stoichiometry but IgG has two bindingsites for FcγRIIIa. Consequently, in a single a-fucosylated antibody thereceptor can bind with high affinity to the binding site formed by theIgG's a-fucosylated glycan and protein core or with low affinity to thebinding site consisting of the fucosylated carbohydrate and the proteincore. It can therefore be concluded that a pool of antibodies with 50%a-fucosylation may consist of a homogeneous population of antibodies inwhich only one of the two N-glycans is fucosylated, or 50% of antibodiesin which both N-glycans are fucosylated while in the other 50% none ofthe N-glycans are fucosylated. It is obvious that such a differentialpartition of a-fucosylation influences the overall affinity to FcγRIIIaand results in a different biological activity. It is thereforemandatory to analyze the biological activity of such an antibodypreparation either directly by employing a biological test system(bioassay) or indirectly by biochemically measuring the rate anddistribution of the a-fucosylation, which yields a more exact result.

The current state-of-the-art glycoanalytics uses N-glycosidase F (PNGaseF) from Flavobacterium meningosepticum to cleave off the N-linkedcarbohydrates with a subsequent MALDI-MS (matrix-assisted laserdesorption ionization mass spectrometry) analysis (according to Papac etal., Glycobiology (1998) 8, 445-454). By employing such a process,however, the linkage information is lost and the determination offucosylation distribution within an antibody preparation is notpossible.

On the other hand, analysis of the complete antibody using ESI-MS(electrospray ionization mass spectrometry) yields complex mass patternsthat do not allow a quantitative interpretation due to the variousmodifications other than fucosylation—like galactosylation, C-terminallysine heterogeneity, deamidation etc.—that may or may not occur in bothsubunits of the homodimeric IgG.

Therefore, there is a need for a new analytical method that eliminatesthe mentioned heterogeneity but maintains the linkage information.

DESCRIPTION OF THE INVENTION

The above described drawbacks are overcome by this invention, whichprovides for methods for detecting the presence or absence of fucoseresidues within a glycosylated antibody. Preferably, the quantity offucose residues and their distribution pattern within an antibody or afragment thereof are determined. The analysis of the distribution offucose residues per Fc molecule in an antibody preparation is also partof this invention. In addition, the present invention can be used forthe determination of cooperative fucosylation in an antibody preparationduring fermentation. Hence, this invention provides for a method thatcloses a gap in antibody analytics. With the knowledge of fucosylationpatterns within an antibody or fragment thereof gained by means of thisnew method, a more accurate prediction of Fc-mediated potency is nowpossible.

Surprisingly, the inventors of the present invention found that Endo S(an enzyme with endoglycosidase activity, originally identified inStreptococcus pyogenes (Collin and Olsen, EMBO J (2001) 20, 3046-3055))cleaves the complex-type glycan moieties from the Fc region of humanIgG, leaving behind just the first GlcNAc residue to which a fucoseresidue might be attached. The hybrid-type carbohydrates that arediscriminated (spared) by Endo S can be quantitatively cleaved at thesame site by Endoglycosidase H (Endo H). The combination of both enzymesthus allows the preparation of a uniformly glycosylated protein thatonly varies by the fucose content. Analysis of such treated Fc fragmentsnot only allows the determination of the fucose content of, but alsodetermination of the distribution of fucose residues within the analyzedantibody pool. These new findings close an analytical gap and may allowa potency estimation of the analyzed antibody in terms of its efficacyin ADCC induction.

Accordingly, the present invention relates to a method for detecting thepresence or absence of fucose residues within a glycosylated antibody ora fragment thereof.

In one embodiment the inventive method comprises the following steps:

a) removal of all heterogeneous saccharide residues from the protein,b) removal of all other heterogeneous residues from the protein,c) subsequent analysis of the protein.

In another embodiment, step c) of said method additionally comprises apurification step prior to analysis. In a specific embodimentpurification is achieved by affinity chromatography or size exclusionchromatography. Affinity chromatography can be performed using forexample Protein A or Protein G.

In one embodiment the protein to be treated and analyzed by the methodof the invention is an antibody or an antibody fragment. Preferably saidantibody is an IgG type antibody. Said antibody fragment is preferablyan Fc fragment, in particular an Fc fragment of an IgG type antibody.

In a specific embodiment the removal of step a) is performed by one ormore enzymes that specifically cleave complex-type or hybrid-typeN-linked carbohydrates. Preferably, these enzymes comprise Endo S andEndo H.

In another specific embodiment the removal of step b) is performed byone or more enzymes. Preferably these enzymes comprise plasmin and/orcarboxypeptidase B.

In a further specific embodiment the analysis of step c) comprisesCE-SDS MW (capillary electrophoresis-sodium dodecyl sulfate molecularweight) analysis, ESI-MS analysis or liquid chromatography-massspectrometry (LC-MS), or a combination thereof.

In a preferred embodiment, step a) of the above described methodcomprises cleavage of the heterogeneous saccharides from thecarbohydrate structures of the protein after the first GlcNAc residue ofsaid structures, thereby leaving the fucose residue attached to theantibody core. This step can be performed with two enzymes thatspecifically cleave complex-type or hybrid-type N-linked carbohydratesthat frequently occur in biotechnologically produced antibodies, forexample Endo S and Endo H.

In a preferred embodiment, step b) of the above described methodcomprises quantitative removal of C-terminal lysine residues of theantibody heavy chain, preferably using an enzyme, said enzyme preferablycomprising carboxypeptidase B.

In another preferred embodiment, step b) of the above described methodcomprises cleavage between the Fab and the Fc fragment of an antibody.Preferably the covalent interchain disulphide bridges within the hingepeptide of the heavy chains are maintained within the Fc-fragment aftercleavage between the Fab and the Fc fragment. Preferably the cleavage isachieved by an enzyme. Preferably such enzyme comprises plasmin.

In a preferred embodiment, step c) of the above described methodcomprises analysis of the treated antibody molecule or Fc fragment byLC-MS without any prior purification steps. Such an analysis normallyyields only three masses that correspond to proteins with twofucosylated glycans, proteins with one fucosylated and one a-fucosylatedglycan, and proteins in which both glycans are a-fucosylated.

In another embodiment, step c) of the above described method comprisespurifying the treated antibody molecule or Fc fragment using standardmethods and analyzing it by ESI-MS analysis. Such an analysis normallyyields only three masses that correspond to proteins with twofucosylated glycans, proteins with one fucosylated and one a-fucosylatedglycan, and proteins in which both glycans are a-fucosylated.

In one embodiment the method of the invention comprises the followingsteps: providing an antibody preparation, optionally isolating the Fcfragment portion of such antibody preparation, removing allheterogeneous saccharide residues from said antibody or Fc fragment withEndo H and Endo S, removing C-terminal lysine residues from saidantibody or Fc fragment with carboxypeptidase B, and analysis of thetreated antibody or Fc fragment by ESI-MS, LC-MS or CE-SDS MW analysis.

In another embodiment said method comprises the following steps:providing an antibody preparation, optionally isolating the Fc fragmentportion of such antibody preparation using plasmin, removing allheterogeneous saccharide residues from said antibody or Fc fragment withEndo H and Endo S, removing C-terminal lysine residues from saidantibody or Fc fragment with carboxypeptidase B, and purification andanalysis of the treated antibody or Fc fragment by ESI-MS, LC-MS orCE-SDS MW analysis.

In yet another embodiment, this invention is directed to kits suitablefor performing an assay which detects the presence or absence of fucoseresidues within a glycoprotein. The kits of this invention comprise allcomponents referred to in the methods described above (e.g. Endo H, EndoS, carboxypeptidase B, plasmin, suitable buffers), instructions settingforth a procedure according to any one of the methods described aboveand a container for the contents of the kit.

Use of Endo S for cleavage of complex-type N-linked oligosaccharides ofa glycoprotein, preferably a glycosylated antibody or a fragmentthereof, is also part of this invention.

DEFINITIONS

Terms are used herein as generally used in the art, unless otherwisedefined in the following:

The term “heterogeneous saccharide” as used herein, includes anymonosaccharide moiety of a glycosylated antibody or antibody fragmentthat is not connected to a fucose residue. Non-limiting examples forheterogeneous saccharides of a glycosylated antibody or antibodyfragment are mannose, sialate, galactose, acetylglucosamine. Generally,heterogeneous saccharides which are removed in step a) of the methodaccording to the invention will be all saccharides other than the firstGlcNAc residue, i.e. the GlcNAc residue attached to an asparagineresidue of the protein, and the fucose residue linked to that firstGlcNAc residue.

The term “heterogeneous residues” as used herein, means any other moietyof a glycosylated antibody or antibody fragment (other than heterogenoussaccharides) that could interfere with the detection of fucose residueswithin said antibody or antibody fragment. Non-limiting examples ofheterogenous residues are various modifications of the glycosylatedantibody or antibody fragment other than fucosylation, such asgalactosylation, C-terminal lysine heterogeneity and deamidation. Theterm “heterogeneous residues” may further include antibody fragmentsthat are not glycosylated, for example the Fab fragment, the scFvfragment and other fragments.

As used herein, the term “antibody” is intended to include wholeantibody molecules, antibody fragments, or fusion proteins that includea region equivalent to the Fc region of an immunoglobulin.

The terms “complex-type oligosaccharide” and “hybrid-typeoligosaccharide” refer to the glycosylation pattern of an antibody orantibody fragment, Non-limiting examples of “complex-typeoligosaccharide” and “hybrid-type oligosaccharide” are shown in FIG. 7.As understood by those skilled in the art, glycoproteins enriched inbisected hybrid-type oligosaccharides typically result fromoverexpression of GnT-III in production cell lines. Exemplary structuresof bisected hybrid-type oligosaccharides are detailed in FIG. 7-III.Glycoproteins enriched in bisected complex type oligosaccharidestypically result from a co-expression of ManII and GnT-III in productioncell lines. Exemplary structures of bisected, complex-typeoligosaccharides are detailed in FIG. 7-IV (Ferrara et al., BiotechnolBioeng (2006) 93, 851-864

Cleavage “after” a sugar residue, as used herein, means cleavage distalto this residue, i.e. cleavage of the sugar bond linking this residuewith the adjacent one towards the outer end of the carbohydratestructure. Cleavage “after the first GlcNAc residue” of an N-linkedglycan means cleavage of the chitobiose core of the oligosaccharide,between the first (i.e. attached to the asparagine residue) and thesecond (i.e., attached to the first) GlcNAc residue.

“Distribution” of fucose residues within an antibody preparation refersto the presence within that preparation of antibody or Fc moleculesdiffering in the number of fucose residues associated with the N-linkedglycans in the Fc region. For example, an IgG molecule has two N-linkedglycans in its Fc region, each of which can have a fucose residue linkedto the first GlcNAc residue of the carbohydrate structure. Thus, in anIgG preparation there might be three different molecular species: IgGwith two, one or no fucose residues associated with the N-linked glycansin the Fc region. The ratio of these different species (i.e. thedistribution of fucose residues per Fc molecule) can be determined bythe method of the invention, in addition to determination of the totalfucose content, i.e. the fraction of fucosylated or a-fucosylatedN-glycans.

The examples below explain the invention in more detail. The examplesare given to enable those skilled in the art to more clearly understandand practice the invention. The present invention, however, is notlimited in scope by the exemplified embodiments, which are intended asillustrations of single aspects of the invention only, and methods whichare functionally equivalent are within the scope of the invention.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of a carbohydrate moiety attached toAsn-297 of human IgG1-Fc. The sugars in bold define the pentasaccharidecore of N-linked glycan structures; the addition of the other sugarresidues is variable. In grey is represented a bisecting GlcNAc residue.

FIG. 2. Deglycosylation of intact Fc fragment of antibodies A (wildtype)and C (glycoengineered) monitored by CE-SDS. Electropherograms ofnon-reduced Fc fragments are shown before and after enzymatic treatment.(A) Fc fragment of antibody C without enzymatic treatment (dashed line)and deglycosylated with PNGase F (dotted line) or Endo S (solid line),(B) Fc fragment of antibody A without enzymatic treatment (dashed line),deglycosylated with PNGase F (dotted line) or deglycosylated with Endo S(solid line).

FIG. 3. Positive-ion MALDI-TOF mass spectra of the N-linkedoligosaccharides released from Fc fragment of antibody C by consecutivetreatment with Endo S and PNGase F or with Endo S and Endo H. (A)Spectrum of glycans released by treatment with Endo S. (B) Spectrum ofEndo S-resistant carbohydrates released by subsequent treatment withPNGase F, resulting in an isolated signal at m/z=1663 (possiblycorresponding to hybrid- or complex-type structures as schematicallydepicted). (C) Spectrum of glycans released by subsequent treatment withthe hybrid-type structure specific enzyme Endo H (hybrid-type structurescorresponding to m/z=1460 released by Endo H treatment are schematicallydepicted).

FIG. 4. Deglycosylation of the Fc fragment of antibody C monitored byCE-SDS MW analysis (A) and positive-ion MALDI-TOF mass spectrometry (B).(A) Overlay of electropherogram of the non-reduced Fc fragment withoutglycosidase treatment (dashed line) and treated with a combination ofEndo S and Endo H (solid line). (B) Mass spectra of the N-linkedoligosaccharides released from the Fc fragment treated with Endo S andEndo H. Hybrid-type structures corresponding to m/z=1460 released byEndo H are schematically depicted.

FIG. 5. ESI-MS spectra of Fc fragments after treatment with Endo S andEndo H. (A) Fc fragments of antibody A, (B) Fc fragments of antibody B,(C) Fc fragments of antibody C. Peak 1: Fc-GlcNAc/GlcNAc, Peak 2:Fc-GlcNAc/GlcNAc+Fuc, Peak 3: Fc-GlcNAc+Fuc/GlcNAc+Fuc.

FIG. 6. Deglycosylation of antibody C monitored by CE-SDS (A) andpositive-ion MALDI-TOF mass spectrometry (B). (A) Electropherograms ofnon-reduced IgG are shown before and after enzymatic treatment: AntibodyC without enzymatic treatment (dashed line) and deglycosylated withPNGase F (dotted line) or combined treatment with Endo S and Endo H(solid line). (B) Mass spectra of the N-linked oligosaccharides releasedfrom entire IgG treated with Endo S and Endo H.

FIG. 7. N-linked oligosaccharide biosynthetic pathway leading tocomplex- or hybrid-type structures. M1: mannosidase I, G1:β1,2-N-acetylglucosaminyltransferase I, G3:β1,4-N-acetylglucosaminyltransferase III, Gt:β1,4-galactosyltransferase.

FIG. 8. ESI-MS spectra of entire IgGs after treatment with Endo S andEndo H. (A) antibody A, (B) antibody D. Peak 1: Fc-GlcNAc/GlcNAc, Peak2: Fc-GlcNAc/GlcNAc+Fuc, Peak 3: Fc-GlcNAc+Fuc/GlcNAc+Fuc.

FIG. 9. LC-MS spectra of entire IgGs after treatment with Endo S andEndo H. (A) antibody A, (B) antibody D. Peak 1: Fc-GlcNAc/GlcNAc, Peak2: Fc-GlcNAc/GlcNAc+Fuc, Peak 3: Fc-GlcNAc+Fuc/GlcNAc+Fuc.

EXAMPLES Example 1 Methods

Generation of Fc from Human IgG

Four different human IgGs with a different content of a-fucosylatedglycans, determined according to Papac et al., 1998 (content inbrackets), were used for analysis of the a-fucosylation distribution:wildtype antibody A (2.12%), glycoengineered antibody B (47.0%),glycoengineered antibody C (69.6%), and glycoengineered antibody D(85%).

The proteins were incubated for 72 hours at 25° C. in 50 mM Tris pH 8.0,150 mM NaCl with 0.42 U plasmin (Roche) per milligram. Cleaved Fc wasseparated from Fab-fragments using a Protein A affinity column (5 mlHiTrap™ Protein A HP column, GE Healthcare) equilibrated and washed (5column volumes (CV)) with buffer A (50 mM Tris pH 8.0, 100 mM glycine,150 mM NaCl). Fc was eluted by a pH-step using buffer B (50 mM Tris pH3.0, 100 mM glycine, 150 mM NaCl). Fractions containing Fc were pooledand neutralized by adding 1:40 (v/v) 2 M Tris pH 8.0. Samples wereconcentrated to a volume of 2.5 ml using ultra concentrators (Vivaspin15R 10'000 MWCO HY, Sartorius) and subsequently applied to a PD-10desalting column (GE Healthcare) equilibrated with 2 mM MOPS pH 7.4, 150mM NaCl, 0.02% (w/v) NaN₃. Purified protein was frozen in liquidnitrogen and stored at −80° C.

Release of N-Linked Oligosaccharides from Human Fc

Different enzymes were used for hydrolyzing the N-linked glycans ofhuman IgG. The N-linked oligosaccharides were cleaved from 1 mg of Fc byincubation with 0.005 U recombinant PNGase F (QAbio, Vista Monte, USA).For release of carbohydrates from Fc using non-tagged Endo S (Genovis),samples were incubated with either a molar ratio of 1:20 of Endo S aloneor in combination with 0.1 U/mg Endo H (QAbio). All reactions wereincubated in 20 mM Tris pH 8.0 at 37° C. for 16 h.

For analyzing carbohydrates spared by Endo S, Fc was purified after EndoS treatment by affinity chromatography using Protein A and subsequentlydigested with either PNGase F or Endo H, as described above.

Release of N-Linked Oligosaccharides from Entire Human IgG

The N-linked glycans of human IgG were released using different enzymes.The N-linked oligosaccharides were cleaved from 1 mg of IgG byincubation with 0.005 U of recombinant PNGase F (QAbio) in 20 mM Tris pH8.0 at 37° C. for 16 h. For release of carbohydrates from IgG usingnon-tagged Endo S (Genovis), samples were applied to a NAP-5 desaltingcolumn (GE Healthcare) equilibrated with 20 mM Tris pH 8.0. Elutedsample was concentrated to a final concentration of 4 mg/ml using ultraconcentrators (Amicon 5′000 MWCO, Millipore) and incubated with a molarratio of 1:7 of Endo S combined with 0.1 U/mg Endo H (QAbio) at 37° C.for 16 h.

Carboxypeptidase B Treatment

To remove heterogenicity caused by C-terminal lysine residues, afterdeglycosylation samples were further incubated with carboxypeptidase B(Roche; 1 mg/ml). Therefore 1 μl carboxypeptidase B per 50 μg human Fcor entire antibody was added to the Endoglycosidase reaction andincubated again for 1 h at 37° C.

MALDI-TOF Mass Spectrometry Analysis of Released Oligosaccharides

Neutral oligosaccharide profiles of the human Fc or entire antibody wereanalyzed by mass spectrometry (Autoflex, Bruker Daltonics GmbH) inpositive ion mode (Papac et al., 1998).

Purification of Deglycosylated Human Fc or Entire Antibody

Fc or entire IgG was separated from enzymes and cleaved carbohydrates byProtein A affinity chromatography using Agilent HPLC 1100 series(Agilent Technologies). Samples were applied to Protein A matrix (Poros20 A; Applied Biosystems) packed in a guard column 2×20 mm C-130B(Upchurch Scientific) equilibrated with buffer A (50 mM Tris, 100 mMglycine, 150 M NaCl, pH 8.0). After washing with 5.5 CV of buffer A,human Fc or entire IgG was eluted by a pH-step using buffer B (50 mMTris, 100 mM glycine, 150 M NaCl, pH 3.0) over 8.3 CV. The fractioncontaining the protein was neutralized by adding 1:40 (v/v) 2 M Tris pH8.0.

The purified protein was subsequently further used for either treatmentwith enzymes to analyze non-cleaved carbohydrates, CE-SDS analysis orESI-MS.

CE-SDS MW Analysis

Deglycosylation was monitored by CE-SDS-MW analysis, using Beckman PA800with UV detection. The buffer of 100 μg of each Protein A purifiedsample was exchanged to 20 mM Tris pH 8.0 using spin concentrators (5000MWCO, Millipore). Non-reduced samples were prepared as described inSDS-MW Analyses Guide using the ProteomeLab SDS-MW Analysis Kit (BeckmanCoulter). The final protein concentration was 1 mg/ml. Samples wereapplied to a preconditioned bare fused silica capillary (50 μm ID×30.2cm). Pre-conditioning and separation were performed according to theinstruction manual.

Sample Preparation for ESI-MS

The buffer of Protein A purified samples was exchanged to 2 mM MOPS pH7.4, 150 mM NaCl, 0.02% (w/v) NaN₃ using spin concentrators (5000 MWCO,Millipore). Proteins were frozen in liquid nitrogen and stored at −80°C.

ESI-MS Analysis of Glycan Structures of Human Fc and Entire IgG byDirect Infusion (Off Line Detection)

Desalting by Size Exclusion Chromatography:

20-50 μg (up to 90 μl) of Fc after treatment of antibody with theproteases plasmin and carboxypeptidase B and with endo-glycosidases EndoS and Endo H, or entire IgG after treatment with Endo S, Endo H andcarboxypeptidase B, were injected onto a Sephadex G25 self-packed ECO SRcolumn (5×250 mm; KronLab) equilibrated with 2% formic acid, 40%acetonitrile at a flow rate of 0.5 ml/min for 30 minutes. The injectedprotein sample was desalted applying an 8 minute isocratic elution with2% formic acid, 40% acetonitrile at a flow rate of 1 ml/min. The elutionof the desalted protein was recorded by UV at 280 nm and the elutingsample (volume about 200-300 μl) was collected in a 1.5 ml reactionvial. An aliquot of the desalted sample was manually filled into a metalcoated glass needle (Proxeon Biosystems Nano ESI-needles, cat#ES387),inserted into the nanospray source of the mass instrument and sprayedinto a ESI-Q-TOF II mass spectrometer from Waters or into a Q-Star Elitemass spectrometer from Applied Biosystems.

MS parameters for direct infusion:

A) of Plasmin-Treated Samples (human Fc) on a Q-TOF II Instrument(Waters)

MS spectra were acquired using a capillary voltage of 1000 V, a conevoltage of 30 V in a mass range from 1000-2000 m/z in positive ion modeusing a source temperature of 80° C. Desolvation temperature was off. MSdata were acquired for approx 2-3 minutes by the respective instrumentsoftware.

B) of Entire Antibody on a MaXis-ESI-MS Instrument (Bruker)

MS spectra were acquired using a NanoMate device as spray interface. Thevalues for data acquisition at the MS instrument were set to 400 Vpp(funnel RF), 120 eV (ISCID energy) and 400 Vpp (Multipol RF) regardingthe transfer parameters, 5.0 eV (ion energy) and 300 m/z (low mass) forthe quadrupol parameters, 15 eV (collision energy) and 3000 Vpp(collision RF) adjusting the collision cell and 800 Vpp, 160 μs fortransfer time and 20 μs prepulse storage at the ion cooler. Data wererecorded at a mass range from 1000-4000 m/z in positive ion mode.

Molar masses of dimeric Fc-fragments and entire antibody, containingdifferent combinations of glycan structures truncated by theendoglycosidases applied, i.e. GlcNAc/GlcNAc, GlcNAc+Fuc/GlcNAc andGlcNAc+Fuc/GlcNAc+Fuc, were determined from the respective m/z patternof the Fc fragment or entire antibody species using an in-housedeveloped software. The relative ratios of the various residuallyglycosylated dimeric Fc fragments or entire antibodies were calculatedwith the same in-house software using the sum of peak areas of the m/zspectrum of a distinct glycosylation variant of the dimeric Fc-fragmentor entire antibody.

ESI-MS Analysis of Glycan Structures of Entire IgG by LC-MS (On LineDetection)

LC-MS was performed on a Dionex HPLC system (Dionex Ultimate 3000)coupled to a Q-TOF II mass spectrometer (Waters). The chromatographicseparation was performed on a ACE C4 column (5 μm particle size, 300 Apore size, 1×30 mm; Advanced Chromatography Technologies). Eluent A was0.1% formic acid, eluent B was 99.9% acetonitrile and 0.1% formic acid.The flow rate was 100 μl/min, the separation was performed at 75° C. and2 μg (10 μl) of an intact antibody sample treated with Endo S and EndoH, but without plasmin treatment, were used.

TABLE 1 Parameters for LC-MS. Time (min.) % B remark 0 25 waste 3 25 3.125 3.5 25 switch to MS 4.0 25 9.0 50 9.5 100 12.5 100 12.6 25 14.9 25switch to waste 15.0 255 stop MS-detection

MS spectra were acquired using a capillary voltage of 2700 V, a conevoltage of 80 V in a mass range from 1000-4000 m/z in positive ion modeusing a source temperature of 100° C. Desolvation temperature was set to200° C. MS data were acquired for approximately 11.4 minutes (gradienttime 3.5 to 14.9 min) by the respective instrument software.

Molar masses of intact antibody (consisting of two heavy chains and twolight chains) containing different combinations of glycan structurestruncated by the endoglycosidases applied, i.e. GlcNAc/GlcNAc,GlcNAc+Fuc/GlcNAc and GlcNAc+Fuc/GlcNAc+Fuc, were determined from therespective m/z pattern of the antibody species using an in-housedeveloped software. The relative ratios of the various residuallyglycosylated intact antibodies were calculated with the same in-housesoftware using the sum of peak areas of the m/z spectrum of a distinctglycosylation variant of the intact antibody.The ratio of non-fucosylated heavy chains was determined by reducing theEndoS and EndoH-treated antibody with TCEP(Tris(2-carboxyethyl)phosphine hydrochloride) and performing an LC-MSanalysis as described before, using the same column type and gradientsetting but some modified parameters for MS data acquisition. MSparameters were the same as described before, but cone voltage was setto 25 V and mass range was from 600-2000 m/z.

Example 2 Results Deglycosylation of Fc

N-Glycosidase F, also known as PNGase F, is a highly specificdeglycosidase that cleaves between the innermost N-acetylglucosamine ofhigh mannose-, hybrid-, and complex-type N-linked oligosaccharides andthe asparagine residue of the glycoprotein to which the glycan isattached (Tarentino et al., 1985). Treatment of the Fc fragments ofantibody A and C with PNGase F according to the instructions of themanufacturer was monitored by CE-SDS. Under these conditions PNGase Fquantitatively removes the glycan moiety of both analyzed samples,resulting in a mobility shift of the main peak from 3.79×10⁻⁵ to3.9×10⁻⁵ (FIG. 2).

Endo S cleaves the chitobiose core of N-linked oligosaccharides, leavingthe first N-acetylglucosamine residue—and an α-fucose residue in case offucosylated carbohydrates—attached to the protein. The CE-analysis of asuch digested glycoengineered sample revealed that approximately 10% ofthe protein were still undigested (FIG. 2 a, Table 2), as demonstratedby a peak with a mobility of 3.84×10⁻⁵. Subsequent analysis by PNGase Ftreatment indicated that the Endo S resistant carbohydrates wereentirely of hybrid structure suggesting specificity of this enzyme forcomplex carbohydrates. This result could be corroborated by thequantitative Endo S digestion of wildtype antibody A which resulted inhomogenously deglycosylated protein (FIG. 2 b).

TABLE 2 Peak area of enzyme-treated Fc fragments evaluated by CE-SDS.Peak area [%] Antibody, enzyme Non-cleaved Cleaved A, no enzyme 99.3 0.7A, PNGase F 1.3 98.7 A, Endo S 1.8 98.2 C, no enzyme 100.0 0.0 C, PNGaseF 0.3 99.7 C, Endo S 10.6 89.4

To confirm this hypothesis, Endo S-treated Fc of antibody C was purifiedby affinity chromatography to remove the enzyme and cleavedcarbohydrates, and subsequently incubated with PNGase F to remove theentire glycan moiety. The hydrolyzed carbohydrates were further analyzedby MALDI TOF MS. The obtained spectra showed that Endo S isdiscriminating (i.e. sparing) either complex- or hybrid-type bisectedstructures that are corresponding to m/z=1663 (FIG. 3 b).

Further experiments were performed to determine whether thediscriminated carbohydrates are complex- or hybrid-type bisectedstructures. After purification by affinity chromatography, the EndoS-treated Fc fragment of antibody C was incubated with PNGase F orEndoglycosidase H (Endo H). Endo H is a recombinant glycosidase thatcleaves within the chitobiose core of high mannose- and hybrid-typeN-linked oligosaccharides of glycoproteins. It is not able to cleavewithin complex structures. MALDI TOF MS spectra showed that thecarbohydrates discriminated by Endo S are cleaved by Endo H, resultingin a main peak of m/z=1460 (FIG. 3 c). These data clearly show that EndoS is not able to release hybrid-type bisected carbohydrates from theasparagine-linked N-acetylglucosamine.

To obtain homogenously deglycosylated material that only varies in itsα-linked fucose content, a combined treatment of the Fc fragment ofantibody C with Endo S and Endo H was performed resulting in a proteinthat was quantitatively deglycosylated after the first GlcNAc residue asobserved by CE-SDS (FIG. 4 a). MALDI-TOF MS analysis showed that thehybrid bisected structures (m/z=1460) are released by combination ofthese two enzymes (FIG. 4 b). To confirm that there is no othercarbohydrate attached to the N-acetylglucosamine with or without anα-linked fucose residue, Endo S- and Endo H-treated Fc fragment ofantibody C was incubated with PNGase F. No MALDI TOF spectra could beobtained after this treatment suggesting that no other carbohydrateswere remaining that cannot be cleaved by Endo S or Endo H (data notshown).

Determination of the Fucose Distribution in a Fc Preparation

To quantify the distribution of the fucose linked to theN-acetylglucosamine residue attached to the Fc, ESI-MS analyses wereperformed. After incubation with Endo S and Endo H before separation byaffinity chromatography, the Fc domains of antibodies A, B and C(generated by plasmin digestion) were treated with carboxypeptidase B toremove heterogeneity introduced by C-terminal lysine.

ESI-MS spectra revealed Fc fragments with either two, one or no fucoselinked to the residual GlcNAc still attached to the protein afterEndoS/EndoH treatment (FIG. 5). Distribution of these three fucosespecies is summarized for the investigated three different IgGs A, B andC (calculated as relative ratio of the sum of peak areas in them/z-spectra). The results correlate well with the fucose contentdetermined by MALDI-TOF MS (Table 3).

TABLE 3 Comparison of the a-fucosylation degree determined by massspectrometry for Fc fragment of antibody A, B and C. MALDI-TOF ESI-MSNon-fuc 2 fucose 1 fucose 0 fucose Non-fuc [%] [%] [%] [%] [%] A 2.12 943 3 4.5 B 47.0 29 41 30 50.5 C 69.6 20 40 40 60.0

Deglycosylation of Entire IgG

For deglycosylation of an entire IgG by combined treatment with Endo Sand Endo H, cleavage conditions had to be optimized. Deglycosylationwith a molar ratio of Endo S to IgG of 1:20, as was used fordeglycosylation of the Fc fragment, was insufficient to deglycosylateentire IgG. Increasing the concentration of Endo S to a molar ratio of1:7 was sufficient to get homogenously deglycosylated material that onlyvaries in its α-linked fucose content observed by CE-SDS (FIG. 6 a).MALDI-TOF analysis showed that the carbohydrates are released bycombined treatment with Endo S and Endo H (FIG. 6 b). Using thisapproach it is possible to analyze the allocation of fucose per IgGwithout separate generation of the Fc-fragment.

Determination of the Fucose Distribution of Entire IgG

Quantification of the distribution of fucose linked to the innermostN-acetylglucosamine residue of N-linked glycans of entire IgGs wasperformed using wildtype antibody A (2.12% a-fucosylation) andglycoengineered antibody D (85.0% a-fucosylation). After combinedtreatment with Endo S and Endo H, both IgGs were incubated withcarboxypeptidase B to remove heterogeneity introduced by C-terminallysine. The antibodies were subsequently purified by affinitychromatography.

Allocation of the core fucose per IgG was determined using two differentmethods. The pattern of the m/z-spectra obtained by ESI-MS off linedetection revealed IgG-species with either two, one or no fucoseattached to the residual GlcNAc after EndoS/EndoH treatment (FIG. 8).Distribution of these three fucose species is summarized for theinvestigated two different IgGs A and D (calculated as relative ratio ofthe sum of peak areas in the m/z-spectra) (Table 4).

LC-MS analyses were also performed to determine the allocation of fucoseper IgG (FIG. 9). For both IgGs, m/z-spectra showed a similar ratio ofspecies with either two, one or no fucose attached as observed in ESI-MSoffline detection, (Table 4). Peak areas below 5% are in the detectionsensitivity of the methods for entire IgG. Ratio for non-fucosylatedheavy chain is presented in Table 4, column Non-fuc [%].

TABLE 4 Comparison of the a-fucosylation degree and fucose allocationdetermined by ESI-MS and LC-MS analyses for antibody A and D. ESI-MSLC-MS 2 fucose 1 fucose 0 fucose Non-fuc 2 fucose 1 fucose 0 fucoseNon-fuc [%] [%] [%] [%] [%] [%] [%] [%] A 92 5 <5 8 94 6 <5 12 D 9 24 6781 10 24 66 80

1.-13. (canceled)
 14. A method for detecting the presence or absence offucose residues within a glycosylated antibody or a fragment thereof,comprising a) providing an antibody preparation comprising aglycosylated antibody or a fragment thereof, b) removing heterogeneoussaccharide residues from the antibody or the fragment thereof byenzymatic means, c) removing other heterogeneous residues from theantibody or the fragment thereof by enzymatic means, and d) analyzingthe antibody or the fragment thereof for the presence or absence offucose residues.
 15. The method of claim 14, further comprising apurification step prior to analyzing the antibody or the fragmentthereof.
 16. The method of claim 14, further comprising determining thequantity of fucose residues and the distribution pattern of fucoseresidues among the molecules within the antibody preparation.
 17. Themethod of claim 14, further comprising determining the distribution offucose residues per Fc molecule in the antibody preparation.
 18. Themethod of claim 14, wherein removing heterogeneous saccharide residuesfrom the antibody or the fragment thereof is performed by one or moreenzymes selected from the group consisting of Endo S and Endo H.
 19. Themethod of claim 14, wherein removing heterogeneous residues from theantibody or the fragment thereof is performed by one or more enzymesselected from the group consisting of plasmin and carboxypeptidase B.20. The method of claim 14, wherein analyzing the antibody or thefragment thereof is performed by an analysis selected from the groupconsisting of LC-MS analysis, CE-SDS MW analysis, and ESI-MS analysis.21. A method for detecting the presence or absence of fucose residueswithin a glycosylated antibody or a fragment thereof, comprising a)providing an antibody preparation comprising a glycosylated antibody, b)optionally isolating the Fc fragment portion of the antibodypreparation, c) removing all heterogeneous saccharide residues from theglycosylated antibody or the Fc fragment with Endo H and Endo S, d)removing C-terminal lysine residues from the antibody or the Fc fragmentwith carboxypeptidase B, and e) analyzing the antibody or the Fcfragment for the presence or absence of fucose residues by LC-MSanalysis, CE-SDS MW analysis, or ESI-MS analysis.
 22. The method ofclaim 21, further comprising a purification step prior to analyzing theantibody or the Fc fragment.
 23. The use of the method of claim 14 orclaim 21 for determining cooperative fucosylation in an antibodypreparation during fermentation.
 24. A kit for use in qualitative andquantitative detection of fucose residues within a peptide, comprisingEndo S and/or Endo H, plasmin and/or carboxypeptidase B, instructionssetting forth a procedure for the qualitative and quantitative detectionof fucose residues within the peptide, and a container for the contentsof the kit.
 25. The use of EndoS for cleaving complex-typeoligosaccharides from a glycoprotein.